摘要
目的 了解百日咳鲍特菌Ⅰ相菌株外膜蛋白 (outermembraneproteins,OMP)生物学活性与IgE介导哮喘的关系。方法 采用去垢剂处理、DEAE 5 2和SephadexG 15 0层析法制备百日咳鲍特菌Ⅰ相菌株OMP及其组分 ,以SDS PAGE估计OMP组分的相对分子质量 (Mr)。采用ELISA检测百日咳全菌死疫苗诱导动物产生的总IgE和卵白蛋白 (ovalbumin ,OVA)特异的IgE量。采用肥大细胞脱颗粒试验观察OMP及其组分促进脱颗粒的作用、ELISA检测培养物上清中组胺水平。采用组胺敏感试验检测OMP及其组分增加小鼠对组胺致死敏感性的作用。结果 从百日咳鲍特菌OMP中分离获得Mr 为 (30、32、38和 6 9)× 10 3的 4个组分。 1～ 10 0 μg mlOMP及其组分均未显示凝集鲎变形细胞溶解物的活性。OVA和百日咳鲍特菌联合免疫豚鼠的血清总IgE和OVA特异性IgE较单用OVA免疫有明显增加 (t=2 .75～ 6 .5 9;P <0 .0 5～ 0 .0 1)。OMP及其Mr30× 10 3、Mr32× 10 3组分有明显的促豚鼠肥大细胞脱颗粒的作用 (t=4 .914～ 9.137,P <0 .0 1) ,但Mr38× 10 3、Mr6 9× 10 3组分无此活性。OMP及其Mr30× 10 3、Mr32× 10 3组分处理的上清中组胺水平明显高于阴性对照组 (t =5 .6 72～9.30 4 ,P <0 .0 1) ,但Mr38× 10 3、Mr6 9× 10 3组分无此活性。结论 百日咳?
ObjectiveTo understand the correlation between the biological activity of out membrane proteins(OMPs) of B.pertussis strain phase Ⅰ and IgE-mediated asthma. MethodsOMPs and its components from B.pertussis phase Ⅰ strain 58001 were prepared by detergent treatment,DEAE-52 and Sephadex G-150 chromatography. The molecular mass of the OMP components were measured by SDS-PAGE. By using limulus lysate assay,the endotoxin activity of the preparations was detected. An ELISA was established to determine the effects of whole dead B.pertussis on inducing total IgE and ovalbumin (OVA)-specific IgE. Mastocyte degranulation test was performed to observe the activities of the OMPs and its components on promoting degranulation in guinea pig mastocyte and the histamine in the supernatant of the cultures was detected by ELISA. Histamine sensitive test was used to demonstrate the effects of the OMPs and its components on increasing histamine lethal sensitivity in mice. ResultsFour components with molecular mass of 30 000,32 000,38 000 and 69 000 from the B.pertussis OMPs were obtained by chromatography. These preparations did not show any activity of coagulating limulus lysate at the concentrations of 1-100μg/ml. When the guinea pigs were co-immunized with OVA and whole dead B.pertussis,the total IgE and OVA-specific IgE in the animals′ sera were significantly increased than those immunized with OVA alone (t=2.75-6.59;P<0.05-0.01). The OMPs and its two components with molecular mass of 30 000 and 32 000 showed remarkably positive inducement on degranulation of guinea pig mastocytes (t= 4.914-9.137,P<0.01) but the other two components did not. The histamine levels in the supernatants treated with the OMPs and its molecular mass of 30 000 and 32 000 components were significantly higher than those of the negative controls (t=5.672-9.304,P<0.01) but molecular mass of 38 000 and 69 000 components were not. ConclusionWe proved the strong IgE adjuvant activity of B.pertussis and the effects of some components from the bacterial OMPs on promoting degranulation of mastocyte and release of histamine in vitro,and increasing hist-amine lethality in vivo,which indicated a close correlation between the infection or vaccination of B.pertussis and IgE-mediated asthma.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第8期606-610,共5页
Chinese Journal of Microbiology and Immunology
基金
浙江省自然科学基金资助项目 (编号 :3 95 0 3 9)
